Rapid label-free visual detection of KRAS mutations using peptide nucleic acid and unmodified gold nanoparticles (original) (raw)
Related papers
High-resolution melting for detecting KRAS mutations in colorectal cancer
Biomedical Reports, 2019
Colorectal cancer is a leading cause of cancer that may metastasize. KRAS gene sequence of exon 2 should be examined for identification of patients that can be treated with anti-EGFR. The aim of the present study was to evaluate the efficacy of high-resolution melting (HRM) to detect KRAS mutations in colorectal cancer (CRC) tumors. The exon 2 of KRAS was amplified from 47 adenocarcinoma CRC tissues. The tumors were subjected to high-resolution melt using quantitative PCR to identify wild-type and mutant subgroups. The results were compared to the mutations detected by next-generation sequences (NGS). The study included 47 patients, with a mean age of 62 years, of whom 24 patients were male. Most of the patients had stage II or stage III tumors. The mean melting temperatures for the wild-type and mutated group at exon 2 were 78.13˚C and 77.87˚C, respectively (P<0.001, 95% CI = 0.11-0.4). The sensitivity and specificity of high-resolution melting were 83.3 and 96.6%, respectively, with a high concordance between the NGS and HRM methods for detecting KRAS mutation in exon 2 (ĸ = 0.816; P=0.625). Thus, HRM could be used as an alternative method for detecting KRAS mutations in colorectal cancer tissue.
Comparison of methodologies for KRAS mutation detection in metastatic colorectal cancer
Cancer Genetics, 2011
Cetuximab and panitumumab are two monoclonal antibodies targeting the epidermal growth factor receptor that have been approved for treatment of metastatic colorectal cancer. Recent clinical trials found an association between KRAS mutation status and resistance to antie epidermal growth factor receptor therapy, leading to the recommendation to perform KRAS mutation analysis before cetuximab or panitumumab treatment. This study was designed to compare and evaluate the efficacy of four different methodologiesdhigh resolution melting, Sanger sequencing, DxS kit, and SNaPshotdfor KRAS mutation detection in a clinical setting. In total, 372 samples from patients with metastatic colorectal cancer were analyzed by high resolution melting and SNaPshot, with 184 of those being further analyzed by Sanger sequencing and 188 with the DxS kit. Sensitivities were compared after consensus findings were determined by the presence of the same result in two of the three methodologies used in each case. The frequency of KRAS codon 12 and 13 mutations in our population was 43.5%, and a discordant finding was observed in 22 samples. Comparing to Sanger sequencing, significantly more consensus mutations were detected by the DxS kit (P Z 0.0139), high resolution melting (P Z 0.0004), and SNaPshot (P Z 0.00001), but no statistically significant differences were found among the three methodologies with higher sensitivity. Keywords KRAS, metastatic colorectal cancer, mutation detection ª 2011 Elsevier Inc. All rights reserved.
Cancer science, 2016
In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the circulating cell-free DNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations by using Luminex method, and in five of 55 patients with tumors containing wild-type KRAS. Curative resection was performed for seven of the 34 patients with primary and circulating cell-free DNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of the seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal gr...
Annals of Surgical Oncology, 2010
Network suggested conducting a KRAS mutations test in metastatic colorectal cancer (mCRC) patients prior to administering therapy that uses anti-epidermal growth factor receptor (EGFR) monoclonal antibody. However, tests of KRAS mutations have been limited when traditional molecular techniques, such as polymerase chain reaction (PCR) combined direct sequencing, are used to obtain and analyze patients' cancer tissues. If the primary tumor or metastatic tissues of patients with mCRC is unavailable, then such analysis will not be feasible. Our laboratory has successfully established a colorimetric membrane array analysis platform that could detect activating KRAS mutations from the peripheral blood of patients with various malignancies.
Nanofluidic Digital PCR for KRAS Mutation Detection and Quantification in Gastrointestinal Cancer
Clinical Chemistry, 2012
BACKGROUND Concomitant quantification of multiple mutant KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) alleles may provide information in addition to that provided by standard mutation-detection procedures. We assessed the feasibility of a nanofluidic digital PCR array platform to detect and quantify KRAS mutations simultaneously in clinically relevant samples. METHODS We assessed 2 groups of patients (colorectal and pancreatic disease): Group 1 consisted of 27 patients with colorectal carcinomas, 14 patients with adenomas, and 5 control individuals; group 2 consisted of 42 patients with pancreatic carcinoma, 4 with adenocarcinomas of the ampulla, and 6 with chronic pancreatitis). Digital PCR was performed with the Digital Array Chip (Fluidigm). RESULTS Nanofluidic digital PCR detected mutant alleles at 0.05% to 0.1%, depending on the variant analyzed. For the colorectal disease group, conventional PCR detected 9 (64%) of 14 adenomas that were positive for KRAS mutants...
British journal of cancer, 2012
KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. We conducted a two-site comparison of two commercial KRAS mutation kits - the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit - and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates ...
Biosensors
In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specif...
Nanotechnology in Colorectal Cancer for Precision Diagnosis and Therapy
Frontiers in nanotechnology, 2021
Colorectal cancer (CRC) is the third most frequently occurring tumor in the human population. CRCs are usually adenocarcinomatous and originate as a polyp on the inner wall of the colon or rectum which may become malignant in the due course of time. Although the therapeutic options of CRC are limited, the early diagnosis of CRC may play an important role in preventive and therapeutic interventions to decrease the mortality rate. The CRC-affected tissues exhibit several molecular markers that may be exploited as the novel strategy to develop newer approaches for the treatment of the disease. Nanotechnology consists of a wide array of innovative and astonishing nanomaterials with both diagnostics and therapeutic potential. Several nanomaterials and nano formulations such as Carbon nanotubes, Dendrimer, Liposomes, Silica Nanoparticles, Gold nanoparticles, Metal-organic frameworks, Core-shell polymeric nano-formulations, Nano-emulsion System, etc can be used to targeted anticancer drug delivery and diagnostic purposes in CRC. The light-sensitive photosensitizer drugs loaded gold and silica nanoparticles can be used to diagnose as well as the killing of CRC cells by the targeted delivery of anticancer drugs to cancer cells. This review is focused on the recent advancement of nanotechnology in the diagnosis and treatment of CRC.
BMC cancer, 2016
KRAS mutation testing is mandatory in the management of metastatic colorectal cancer prior to treatment with anti-EGFR antibodies as patients whose tumors express mutant KRAS do not benefit from these agents. Although the U.S. Food and Drug Administration has recently approved two in-vitro diagnostics kits for determination of KRAS status, there is generally no consensus on the preferred method and new tests are continuously being developed. Most of these techniques focus on the hotspot mutations at codons 12 and 13 of the KRAS gene. We describe a two-step approach to KRAS codon 12/13 mutation testing involving high resolution melting analysis (HRM) followed by pyrosequencing using the Therascreen KRAS Pyro kit (Qiagen) of only those samples that are not clearly identified as KRAS wildtype or mutant by HRM. First, we determined KRAS status in a panel of 61 colorectal cancer samples using both methods to compare technical performance and concordance of results. Subsequently, we evalu...